A practical set of HPLC methods was developed for the separation and determination of the eggplant steroidal glycoalkaloids, solanine, chaconine, solasonine, solamargine, and their aglycones, solasodine and solanidine. A gradient method was initially developed, but proved to be neither robust nor practical. Three separate isocratic methods using acetonitrile and ammonium dihydrogen phosphate were developed and shown to be more repeatable, less subject to fluctuations in mobile phase composition, and less time consuming. The effect of adjusting buffer pH, column temperature, and buffer type (triethylammonium phosphate vs. ammonium dihydrogen phosphate) were evaluated. It was also discovered that, by addition of 10% methanol to the acetonitrile portion of the mobile phase, more control over the separations was possible. The use of methanol as a mobile phase entrainer greatly improved separations in some cases and its effectiveness was also dependent upon column temperature. Assessments of the method recovery, limit of detection, and limit of quantitation were made using extracts from S. melongena and S. linnaeanum.
The colorimetric and fluorometric detection of D-fructose was achieved by employing a two component sensing system composed of an arylboronic acid as the host molecule and a pH sensitive spirocyclic rhodamine dye as the indicator molecule.Rhodamine dyes are well-known fluorophores with remarkable photophysical properties, such as long absorption and emission wavelengths, high fluorescence quantum yields and large absorption coefficients. The unique ring opening equilibrium of their spirocyclic derivatives has been widely utilized in the design of turn-on type fluorescent sensors.1 The rhodamine spirolactam can exist in two isomeric forms, depending on the medium pH (Scheme 1). The rhodamine spirolactam in its closed form is non-fluorescent at neutral pH; however, the structure undergoes a change from the spirolactam to an open ring amide at lower pH (less than 5.0), resulting in a visibly colorful and highly fluorescent rhodamine amide.
2The ability of rhodamine based dyes to change their color and fluorescence in response to pH changes has been extensively exploited in monitoring the pH changes in various environments (i.e. in solution, atmosphere and living cells).2,3 Relying on their promising chemical and photophysical properties we envisioned that pH sensitive rhodamine dyes could serve as pH indicators in a saccharide sensing system, based on a boronic-saccharide affinity pair. It is well known that boronic acids show high affinities for diols. 4 A boronic acid reversibly forms a cyclic boronate ester with diols (Scheme 2), and in aqueous media the formation of the boronate ester is accompanied by an increase in hydronium ion concentration (i.e. lowered pH).
4It was our aim to develop a general sensing strategy that allows one to monitor the binding of a target saccharide to simple unmodified boronic acids both colorimetrically and fluorometrically. Considering the high sensitivity of spirocyclic rhodamine dyes to pH changes we designed a two component sensing system that employs an aryl boronic acid molecule as the host and a rhodamine B spirolactam dye as the signal reporter.Practical examples of sensors that are based on this sensing principle have been reported recently. 5,6 The majority of the reported studies, however, have been focused on the colorimetric responses of the indicator dyes. 5a,b Examples of detection methods that employ pH sensitive fluorophore indicators are quite rare. 6 In general, fluorometric detection methods are more sensitive than colorimetric detection methods in terms of both the detection limit and signal magnitude. In this regard, a method based on fluorometric detection might be well suited to meet the need for the highly efficient analysis of saccharide-containing samples.
Solid-phase microextraction (SPME), followed by on-fiber derivatization was investigated for the analysis of the steroidal glycoalkaloid aglycones, solasodine and solanidine. The aglycones were first extracted by direct immersion of the SPME fiber in the sample medium and then derivatized on the fiber in a separate step using 1-(trimethylsilyl)imidazole (TMSI). The derivatized compounds were then desorbed from the SPME fiber and detected by GC-MS. Polydimethylsiloxane/Divinylbenzene (PDMS-DVB), Carboxen/Polydimethylsiloxane (CAR-PDMS), and Carbowax/Divinylbenzene (CW-DVB) fibers were employed with the CW-DVB fibers being the most successful, as expected. Closed-end capillary tubes were used to hold the extraction media. Both aglycones were successfully extracted, derivatized, and detected by GC-MS. Solasodine always required derivatization, but solanidine did not.The same method was successfully applied to cholesterol so that it could be used as an internal standard. Also, using the closed-end capillary tubes, a two-phase extraction system was also investigated, whereby the fiber was only exposed to the phase in which it was presumed to be less damaged. However, in all cases, fiber degradation was significant, preventing the use of extended extraction times and limiting reuse of the fibers. However, the results represent a first look into the feasibility of the method. With the development of more suitable SPME phases, this method could potentially provide a complementary route for routine determinations of glycoalkaloids for both research and food quality control.
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